The goal of the proposed research is to gain an understanding of the molecular basis of visual excitation in mammalian retinal rod cells. The conformation of rhodopsin in disc membranes and light-induced structural changes will be investigated by spectroscopic, electron microscopic, and biochemical techniques. We propose to carry out the following studies: (1) Fluorescence energy transfer will be used as a spectroscopic ruler to map the architecture of rhodopsin in situ. Distances between various sulfhydryl groups, the carbohydrate units, the site labeled by transglutaminase and retinal will be measured. The location of retinal relative to the intra-disc membrane surface will be determined by using terbium chelates as energy donors. Energy transfer experiments will also be carried out to ascertain whether rhodopsin is monomeric or oligomeric in the disc membrane, and whether the degree of association changes on illumination. (2) disc membranes bound to beads and as vesicles will be enzymatically-labeled using transglutaminase and galactosyl transferase and then subjected to specific cleavage by subtilisin to determine whether the carbohydrate units of rhodopsin are located on the intradisc or extradisc side of the membrane. (3) The membrane sidedness of several sites on rhodopsin will be investigated by electron microscopic localization of ferritin-antibody and ferritin-avidin conjugates. Cathepsins from pigment epithelium lysosomes will be used to cleave the F1 and F2 fragments of rhodopsin for structural analysis and use as antigens. (4) Neutron diffraction studies of intact retinas containing deuterated retinal will be carried out to determine the location of the chromophoric site in rhodopsin by the difference Fourier technique. (5) Rod outer segment proteins such as the cyclic GMP phosphodiesterase and the GTPase will be labeled with fluorescent reagents to investigate their interactions with rhodopsin.